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Abstract

Circulating endothelial progenitor cells (EPCs) are hypothesized to participate in postnatal vasculogenesis and are currently being infused into patients with cardiovascular disease. However, methods for identification of EPCs vary and do not employ standard principles used to define other stem/progenitor cells. EPCs are commonly identified by the cell surface antigen expression of CD34, CD133, and VEGFR-2 or via counting in a commercially available kit that identifies colony forming unit-endothelial cells (CFU-EC). CFU-EC have limited proliferative capacity and may be contaminated with hematopoietic cells. Recently, highly clonogenic endothelial colony forming cells (ECFC) were identified in human peripheral blood and can be organized in a hierarchy of progenitor stages based on proliferative potential. In this study, CFU-EC and ECFC isolated from peripheral blood were examined for vessel-forming ability in vivo, endothelial and hematopoietic phenotype via confocal microscopy, and clonogenic potential in progenitor assays. In an in vivo transplantation model, ECFC form perfused vessels while CFU-EC show no vessel forming ability. Both CFU-EC and ECFC express endothelial cell surface antigens and take up acetylated-LDL. However, only CFU-EC express hematopoietic antigens and macrophage specific CD115. CFU-EC but not ECFC also display nonspecific esterase activity and ingest bacteria, which are hallmarks of macrophage function. In progenitor assays, ECFC give rise to numerous secondary ECFC colonies, whereas CFU-EC form no secondary endothelial colonies. When replated in specific conditions, however, CFU-EC give rise to secondary hematopoietic granulocyte-macrophage colonies. Moreover, in polycythemia vera patients harboring a JAK2 mutation in hematopoietic stem cell clones, all CFU-EC display the abnormal genotype while ECFC do not. These data indicate that CFU-EC are hematopoietic derived macrophages with no ability to form secondary colonies or perfused vessels in vivo and are not clonally related to ECFC. In contrast, ECFC are clonogenic EPCs with vessel forming activity in vivo. Thus, these studies establish that CFU-EC are not EPCs and their role in angiogenesis must be reexamined prior to further clinical trials.